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Abstract:

A novel compound having an excellent NFκB inhibitory effect is
provided and specifically disclosed is a compound represented by the
following formula (1) or a salt thereof:
wherein, R1 represents a hydrogen atom, a lower alkyl group, a lower
alkyloxy group, or a halogen atom; R2 represents a hydroxyl group,
or a lower alkyloxy group; R3 represents a hydrogen atom, a lower
alkyl group, or a halogen atom; R4 represents a hydrogen atom or a
lower alkyloxy group; R5 represents a hydrogen atom, a lower
alkyloxy group, a halogen atom, a hydroxyl group, or a methylenedioxy
group formed together with R6 or an isopropylidenedioxy group formed
together with R6; R6 represents a hydrogen atom, a lower
alkyloxy group, or a methylenedioxy group formed together with R5 or
an isopropylidenedioxy group formed together with R5; R7
represents a hydrogen atom or a lower alkyl group; and R8 represents
a hydrogen atom, a hydroxyl group, an amino group, or a lower
alkylcarbonyloxy group (excluding the case where R1, R3,
R4 and R7 are hydrogen atoms, R2 and R8 are hydroxyl
groups, and R5 and R6 are methoxy groups).
##STR00001##

Claims:

1. A compound represented by formula (1) or a salt thereof: ##STR00015##
wherein R1 represents a hydrogen atom, a lower alkyl group, a lower
alkyloxy group, or a halogen atom; R2 represents a hydroxyl group,
or a lower alkyloxy group; R3 represents a hydrogen atom, a lower
alkyl group, or a halogen atom; R4 represents a hydrogen atom or a
lower alkyloxy group; R5 represents a hydrogen atom, a lower
alkyloxy group, a halogen atom, a hydroxyl group, or a methylenedioxy
group formed together with R6, or an isopropylidenedioxy group
formed together with R6; R6 represents a hydrogen atom, a lower
alkyloxy group, or a methylenedioxy group formed together with R5,
or an isopropylidenedioxy group formed together with R5; R7
represents a hydrogen atom or a lower alkyl group; and R8 represents
a hydrogen atom, a hydroxyl group, an amino group, or a lower
alkylcarbonyloxy group; provided that the following cases are excluded:
(A) the case where R1, R3, R4, and R7 are hydrogen
atoms, R2 and R8 are hydroxyl groups, and R5 and R6
are methoxy groups; (B) the case where R1, R3, R4, and
R7 are hydrogen atoms, R2, R5, and R6 are methoxy
groups, and R8 is a hydroxyl group; (C) the case where R1,
R3, R4, R7, and R8 are hydrogen atoms and R2,
R5, and R6 are methoxy groups; (D) the case where R1,
R3, R4, and R7 are hydrogen atoms, R2, R5, and
R8 are hydroxyl groups, and R6 is a methoxy group; (E) the case
where R1, R3, R4, and R7 are hydrogen atoms, R2
and R6 are methoxy groups, and R5 and R8 are hydroxyl
groups; (F) the case where R1, R3, R4, R7, and
R8 are hydrogen atoms, R2 and R6 are methoxy groups, and
R5 is a hydroxyl group; (G) the case where R1, R2,
R5, and R6 are methoxy groups and R3, R4, R7,
and R8 are hydrogen atoms; (H) the case where R1, R2,
R4, and R5 are methoxy groups and R3, R6, R7,
and R8 are hydrogen atoms; (I) the case where R1, R2,
R5, and R6 are methoxy groups, R3, R4, and R7
are hydrogen atoms, and R8 is a hydroxyl group; (J) the case where
R1 and R2 are methoxy groups, R3, R4, R7, and
R8 are hydrogen atoms, and R5 is a hydroxyl group; (K) the case
where R1, R2, and R5 are methoxy groups, and R3,
R4, R6, R7, and R8 are hydrogen atoms; (L) the case
where R1, R2, and R5 are methoxy groups, R3, R4,
R6, and R7 are hydrogen atoms, and R8 is a hydroxyl group;
(M) the case where R1, R2, and R5 are methoxy groups,
R3, R4, R6, and R8 are hydrogen atoms, and R7 is
a methyl group; (N) the case where R1, R2, and R5 are
methoxy groups, R3, R4, and R6 are hydrogen atoms, R7
is a methyl group, and R8 is a hydroxyl group; (O) the case where
R1, R2, and R6 are methoxy groups, R3, R4,
R7, and R8 are hydrogen atoms, and R5 is a hydroxyl group;
(P) the case where R1, R2, and R4 are methoxy groups,
R3, R6, R7, and R8 are hydrogen atoms, and R5 is
a hydroxyl group; (Q) the case where R1, R5, and R6 are
methoxy groups, R2 and R8 are hydroxyl groups, and R3,
R4, and R7 are hydrogen atoms; (R) the case where R1 is an
isopropyloxy group, R2 and R5 are methoxy groups, and R3,
R4, R6, R7 and R8 are hydrogen atoms; (S) the case
where R1 and R5 are methoxy groups, R2 is an isopropyloxy
group, and R3, R4, R6, R7 and R8 are hydrogen
atoms; (T) the case where R1 and R2 are methoxy groups,
R3, R4, R6, R7, and R8 are hydrogen atoms, and
R5 is an isopropyloxy group; (U) the case where R1 and R5
are methoxy groups, R2 is a hydroxyl group, and R3, R4,
R6, R7, and R8 are hydrogen atoms; (V) the case where
R1 and R2 are methoxy groups, and R3, R4, R5,
R6, R7, and R8 are hydrogen atoms; (W) the case where
R1, R4, R7, and R8 are hydrogen atoms, and R2,
R3, R5, and R6 are methoxy groups; (X) the case where
R1, R3, R4, and R7 are hydrogen atoms, R2,
R5, and R6 are methoxy groups, and R8 is an acetoxy group;
(Y) the case where R1, R4, and R8 are hydrogen atoms,
R2 and R6 are methoxy groups, R3 and R5 are hydroxyl
group, and R7 is a methyl group; (Z) the case where R1,
R4, R7, and R8 are hydrogen atoms, R2 is a hydroxyl
group, and R3, R5, and R6 are methoxy groups; (AA) the
case where R1, R4, and R7 are hydrogen atoms, R2 and
R8 are hydroxyl groups, and R3, R5, and R6 are
methoxy groups; (AB) the case where R1, R4, R7, and
R8 are hydrogen atoms, R2 and R5 are hydroxyl groups, and
R3 and R6 are methoxy groups; (AC) the case where R1,
R4 and R7, are hydrogen atoms, R2, R5, and R8
are hydroxyl groups, and R3 and R6 are methoxy groups; (AD) the
case where R1, R2, and R5 are methoxy groups, R3,
R4, R7, and R8 are hydrogen atoms, and R6 is a
hydroxyl group; (AE) the case where R1, R3, R4, and
R8 are hydrogen atoms, R2, R5, and R6 are methoxy
groups, and R7 is a methyl group; (AF) the case where R1,
R2, R5, and R6 are methoxy groups, R3, R4, and
R7 are hydrogen atoms, and R8 is an acetoxy group; and (AG) the
case where R1, R2, and R5 are methoxy groups, R3,
R4, R6, and R7 are hydrogen atoms, and R8 is an
acetoxy group.

2. The compound or salt of claim 1, wherein R1 represents a hydrogen
atom, an alkyl group with a carbon number of 1 to 6, an alkyloxy group
with a carbon number of 1 to 6, or a halogen atom; R2 represents a
hydroxyl group, or an alkyloxy group with a carbon number of 1 to 6;
R3 represents a hydrogen atom, an alkyl group with a carbon number
of 1 to 6, or a halogen atom; R4 represents a hydrogen atom or an
alkyloxy group with a carbon number of 1 to 6; R5 represents a
hydrogen atom, an alkyloxy group with a carbon number of 1 to 6, a
halogen atom, a hydroxyl group, or a methylenedioxy group formed together
with R6, or an isopropylidenedioxy group formed together with
R6; R6 represents a hydrogen atom, an alkyloxy group with a
carbon number of 1 to 6, or a methylenedioxy group formed together with
R5, or an isopropylidenedioxy group formed together with R5;
R7 represents a hydrogen atom or an alkyl group with a carbon number
of 1 to 6; and R8 represents a hydrogen atom, a hydroxyl group, an
amino group, or an alkylcarbonyloxy group with a carbon number of 1 to 6;
provided that the following cases are excluded: (A) the case where
R1, R3, R4, and R7 are hydrogen atoms, R2 and
R8 are hydroxyl groups, and R5 and R6 are methoxy groups;
(B) the case where R1, R3, R4, and R7 are hydrogen
atoms, R2, R5, and R6 are methoxy groups, and R8 is a
hydroxyl group; (C) the case where R1, R3, R4, R7,
and R8 are hydrogen atoms and R2, R5, and R6 are
methoxy groups; (D) the case where R1, R3, R4, and R7
are hydrogen atoms, R2, R5, and R8 are hydroxyl groups,
and R6 is a methoxy group; (E) the case where R1, R3,
R4, and R7 are hydrogen atoms, R2 and R6 are methoxy
groups, and R5 and R8 are hydroxyl groups; (F) the case where
R1, R3, R4, R7, and R8 are hydrogen atoms,
R2 and R6 are methoxy groups, and R5 is a hydroxyl group;
(G) the case where R1, R2, R5, and R6 are methoxy
groups and R3, R4, R7, and R8 are hydrogen atoms; (G)
the case where R1, R2, R4, and R5 are methoxy groups
and R3, R6, R7, and R8 are hydrogen atoms; (H) the
case where R1, R2, R5, and R6 are methoxy groups,
R3, R4, and R7 are hydrogen atoms, and R8 is a
hydroxyl group; (I) the case where R1 and R2 are methoxy
groups, R3, R4, R6, R7, and R8 are hydrogen
atoms, and R5 is a hydroxyl group; (J) the case where R1,
R2, and R5 are methoxy groups, and R3, R4, R6,
R7, and R8 are hydrogen atoms; (K) the case where R1,
R2, and R5 are methoxy groups, R3, R4, R6, and
R7 are hydrogen atoms, and R8 is a hydroxyl group; (L) the case
where R1, R2, and R5 are methoxy groups, R3, R4,
R6, and R8 are hydrogen atoms, and R7 is a methyl group;
(M) the case where R1, R2, and R5 are methoxy groups,
R3, R4, and R6 are hydrogen atoms, R7 is a methyl
group, and R8 is a hydroxyl group; (N) the case where R1,
R2, and R6 are methoxy groups, R3, R4, R7, and
R8 are hydrogen atoms, and R5 is a hydroxyl group; (O) the case
where R1, R2, and R4 are methoxy groups, R3, R6,
R7, and R8 are hydrogen atoms, and R5 is a hydroxyl group;
(P) the case where R1, R5, and R6 are methoxy groups,
R2 and R8 are hydroxyl groups, and R3, R4, and
R7 are hydrogen atoms; (Q) the case where R1 is an isopropyloxy
group, R2 and R5 are methoxy groups, and R3, R4,
R6, R7 and R8 are hydrogen atoms; (R) the case where
R1 and R5 are methoxy groups, R2 is an isopropyloxy group,
and R3, R4, R6, R7 and R8 are hydrogen atoms;
(S) the case where R1 and R2 are methoxy groups, R3,
R4, R6, R7, and R8 are hydrogen atoms, and R5 is
an isopropyloxy group; (T) the case where R1 and R5 are methoxy
groups, R2 is a hydroxyl group, and R3, R4, R6,
R7, and R8 are hydrogen atoms; (U) the case where R1 and
R2 are methoxy groups, and R3, R4, R5, R6,
R7, and R8 are hydrogen atoms; (V) R1, R4, R7,
and R8 are hydrogen atoms, and R2, R3, R5, and
R6 are methoxy groups; (W) the case where R1, R3, R4,
and R7 are hydrogen atoms, R2, R5, and R6 are methoxy
groups, and R8 is an acetoxy group; (X) the case where R1,
R4, and R8 are hydrogen atoms, R2 and R6 are methoxy
groups, R3 and R5 are hydroxyl group, and R7 is a methyl
group; (Y) the case where R1, R4, R7, and R8 are
hydrogen atoms, R2 is a hydroxyl group, and R3, R5, and
R6 are methoxy groups; (Z) the case where R1, R4, and
R7 are hydrogen atoms, R2 and R8 are hydroxyl groups, and
R3, R5, and R6 are methoxy groups; (AA) the case where
R1, R4, R7, and R8 are hydrogen atoms, R2 and
R5 are hydroxyl groups, and R3 and R6 are methoxy groups;
(AB) the case where R1, R4 and R7, are hydrogen atoms,
R2, R5, and R8 are hydroxyl groups, and R3 and
R6 are methoxy groups; (AC) the case where R1, R2, and
R5 are methoxy groups, R3, R4, R7, and R8 are
hydrogen atoms, and R6 is a hydroxyl group; (AD) the case where
R1, R3, R4, and R8 are hydrogen atoms, R2,
R5, and R6 are methoxy groups, and R7 is a methyl group;
(AE) the case where R1, R2, R5, and R6 are methoxy
groups, R3, R4, and R7 are hydrogen atoms, and R8 is
an acetoxy group; and (AF) the case where R1, R2, and R5
are methoxy groups, R3, R4, R6, and R7 are hydrogen
atoms, and R8 is an acetoxy group.

3. The compound or salt of claim 1, wherein R1 represents a hydrogen
atom, a methyl group, a methoxy group, a chlorine atom, or a fluorine
atom; R2 represents a hydroxyl group, or a methoxy group; R3
represents a hydrogen atom, a methyl group, a chlorine atom, or a
fluorine atom; R4 represents a hydrogen atom or a methoxy group;
R5 represents a hydrogen atom, a methoxy group, an ethoxy group, a
fluorine atom, a hydroxyl group, or a methylenedioxy group formed
together with R6, or an isopropylidenedioxy group formed together
with R6; R6 represents a hydrogen atom, a methoxy group, an
ethoxy group, or a methylenedioxy group formed together with R5, or
an isopropylidenedioxy group formed together with R5; R7
represents a hydrogen atom or a methyl group; and R8 represents a
hydrogen atom, a hydroxyl group, an amino group, or an acetoxy group;
provided that the following cases are excluded: (A) the case where
R1, R3, R4, and R7 are hydrogen atoms, R2 and
R8 are hydroxyl groups, and R5 and R6 are methoxy groups;
(B) the case where R1, R3, R4, and R7 are hydrogen
atoms, R2, R5, and R6 are methoxy groups, and R8 is a
hydroxyl group; (C) the case where R1, R3, R4, R7,
and R8 are hydrogen atoms and R2, R5, and R6 are
methoxy groups; (D) the case where R1, R3, R4, and R7
are hydrogen atoms, R2, R5, and R8 are hydroxyl groups,
and R6 is a methoxy group; (E) the case where R1, R3,
R4, and R7 are hydrogen atoms, R2 and R6 are methoxy
groups, and R5 and R8 are hydroxyl groups; (F) the case where
R1, R3, R4, R7, and R8 are hydrogen atoms,
R2 and R6 are methoxy groups, and R5 is a hydroxyl group;
(G) the case where R1, R2, R5, and R6 are methoxy
groups and R3, R4, R7, and R8 are hydrogen atoms; (H)
the case where R1, R2, R4, and R5 are methoxy groups
and R3, R6, R7, and R8 are hydrogen atoms; (I) the
case where R1, R2, R5, and R6 are methoxy groups,
R3, R4, and R7 are hydrogen atoms, and R8 is a
hydroxyl group; (J) the case where R1 and R2 are methoxy
groups, R3, R4, R6, R7, and R8 are hydrogen
atoms, and R5 is a hydroxyl group; (K) the case where R1,
R2, and R5 are methoxy groups, and R3, R4, R6,
R7, and R8 are hydrogen atoms; (L) the case where R1,
R2, and R5 are methoxy groups, R3, R4, R6, and
R7 are hydrogen atoms, and R8 is a hydroxyl group; (M) the case
where R1, R2, and R5 are methoxy groups, R3, R4,
R6, and R8 are hydrogen atoms, and R7 is a methyl group;
(N) the case where R1, R2, and R5 are methoxy groups,
R3, R4, and R6 are hydrogen atoms, R7 is a methyl
group, and R8 is a hydroxyl group; (O) the case where R1,
R2, and R6 are methoxy groups, R3, R4, R7, and
R8 are hydrogen atoms, and R5 is a hydroxyl group; (P) the case
where R1, R2, and R4 are methoxy groups, R3, R6,
R7, and R8 are hydrogen atoms, and R5 is a hydroxyl group;
(Q) the case where R1, R5, and R6 are methoxy groups,
R2 and R8 are hydroxyl groups, and R3, R4, and
R7 are hydrogen atoms; (R) the case where R1 is an isopropyloxy
group, R2 and R5 are methoxy groups, and R3, R4,
R6, R7 and R8 are hydrogen atoms; (S) the case where
R1 and R5 are methoxy groups, R2 is an isopropyloxy group,
and R3, R4, R6, R7 and R8 are hydrogen atoms;
(T) the case where R1 and R2 are methoxy groups, R3,
R4, R6, R7, and R8 are hydrogen atoms, and R5 is
an isopropyloxy group; (U) the case where R1 and R5 are methoxy
groups, R2 is a hydroxyl group, and R3, R4, R6,
R7, and R8 are hydrogen atoms; (V) the case where R1 and
R2 are methoxy groups, and R3, R4, R5, R6,
R7, and R8 are hydrogen atoms; (W) the case where R1,
R4, R7, and R8 are hydrogen atoms, and R2, R3,
R5, and R6 are methoxy groups; (X) the case where R1,
R3, R4, and R7 are hydrogen atoms, R2, R5, and
R6 are methoxy groups, and R8 is an acetoxy group; (Y) the case
where R1, R4, and R8 are hydrogen atoms, R2 and
R6 are methoxy groups, R3 and R5 are hydroxyl group, and
R7 is a methyl group; (Z) the case where R1, R4, R7,
and R8 are hydrogen atoms, R2 is a hydroxyl group, and R3,
R5, and R6 are methoxy groups; (AA) the case where R1,
R4, and R7 are hydrogen atoms, R2 and R8 are hydroxyl
groups, and R3, R5, and R6 are methoxy groups; (AB) the
case where R1, R4, R7, and R8 are hydrogen atoms,
R2 and R5 are hydroxyl groups, and R3 and R6 are
methoxy groups; (AC) the case where R1, R4 and R7, are
hydrogen atoms, R2, R5, and R8 are hydroxyl groups, and
R3 and R6 are methoxy groups; (AD) the case where R1,
R2, and R5 are methoxy groups, R3, R4, R7, and
R8 are hydrogen atoms, and R6 is a hydroxyl group; (AE) the
case where R1, R3, R4, and R8 are hydrogen atoms,
R2, R5, and R6 are methoxy groups, and R7 is a methyl
group; (AF) the case where R1, R2, R5, and R6 are
methoxy groups, R3, R4, and R7 are hydrogen atoms, and
R8 is an acetoxy group; and (AG) the case where R1, R2,
and R5 are methoxy groups, R3, R4, R6, and R7
are hydrogen atoms, and R8 is an acetoxy group.

4. The compound or salt of claim 1, wherein the compound represented by
formula (1) has a conformation represented by formula (2): ##STR00016##

5. The compound or salt of claim 4, wherein R8 in the formula (2) is
a hydrogen atom.

7. A medicine, comprising the compound or salt of claim 1 as an active
ingredient.

8. An NFκB inhibitor, comprising the compound or salt of claim 1 as
an active ingredient.

9. A preventive or therapeutic agent, comprising the compound or salt of
claim 1 as an active ingredient, wherein the agent is suitable for
treating or preventing a disease associated with accelerated NFκB
activity.

10. An anticancer agent, comprising the compound or salt of claim 1 as an
active ingredient.

11. A preventive or therapeutic agent for inflammatory disease,
comprising the compound or salt of claim 1 as an active ingredient,
wherein the agent is suitable for treating or preventing inflammatory
disease.

12. A pharmaceutical composition, comprising: the compound or salt of
claim 1; and a pharmaceutically acceptable carrier.

13-15. (canceled)

16. A method for preventing or treating a disease associated with
accelerated NFκB activity, comprising administering to a subject in
need thereof, an effective amount of the compound or salt of claim 1.

17. A method for preventing or treating cancer, comprising administering
to a subject in need thereof, an effective amount of the compound or salt
of claim 1.

18. A method for preventing or treating inflammatory disease, comprising
administering to a subject in need thereof, an effective amount of the
compound or salt of claim 1.

19. A medicine, comprising the compound or salt of claim 6 as an active
ingredient.

20. An NFκB inhibitor, comprising the compound or salt of claim 6
as an active ingredient.

21. A preventive or therapeutic agent, comprising the compound or salt of
claim 6 as an active ingredient, wherein the agent is suitable for
treating or preventing a disease associated with accelerated NFκB
activity.

22. An anticancer agent, comprising the compound or salt of claim 6 as an
active ingredient.

23. A preventive or therapeutic agent for inflammatory disease,
comprising the compound or salt of claim 6 as an active ingredient,
wherein the agent is suitable for treating or preventing inflammatory
disease.

24. A pharmaceutical composition, comprising: the compound or salt of
claim 6; and a pharmaceutically acceptable carrier.

25. A method for preventing or treating a disease associated with
accelerated NFκB activity, comprising administering to a subject in
need thereof, an effective amount of the compound or salt of claim 6.

26. A method for preventing or treating cancer, comprising administering
to a subject in need thereof, an effective amount of the compound or salt
of claim 6.

27. A method for preventing or treating inflammatory disease, comprising
administering to a subject in need thereof, an effective amount of the
compound or salt of claim 6.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a Nuclear Factor-κB
(hereinafter, may be referred to as NFκB) inhibitor. In more
detail, the present invention relates to a novel phenanthroindolizidine
alkaloid compound or a salt thereof inhibiting NFκB, and a medicine
containing the same.

BACKGROUND ART

[0002] NFκB exists as a dimer formed by various combinations of p50,
p65/RelA, c-Rel, Rel-B, and p52, all of which are members of the
NFκB family. Among them, the most well-known dimer is a heterodimer
composed of a 50 kDa subunit (p50) and a 65 kDa subunit (p65).

[0003] Usually, this heterodimer is present in an inactive state in
cytoplasmas through binding to an inhibitor of NFκB (IκB).
However, once the cells are stimulated by inflammatory cytokines, cell
growth factors, and the like, IκB kinase is activated via the AKT
signal transduction pathway and the like, leading to phosphorylation of
IκB. The phosphorylated IκB is ubiquitinated and then
decomposed by proteasome. As a result, NFκB is detached from
IκB and migrate into the nucleus, where it binds to the NFκB
responsive element to activate transcription of various target genes.

[0004] The target genes include many genes associated with inflammation
and immune response (Non Patent Document 1), and the activation of
NFκB is known to be associated with diseases such as rheumatoid
arthritis, osteoarthritis, inflammatory bowel disease, atopic dermatitis,
and asthma (Non Patent Document 2).

[0005] Also, various viruses such as HIV are known to activate NFκB
in host cells, from which NFκB is considered to contribute to viral
infection (Non Patent Documents 3 and 4).

[0006] Furthermore, recently, NFκB is known to be often
constitutively activated in various tumor, and thus it is considered that
NFκB may possibly be involved also in the induction of expression
of various genes associated with the progression of cancer, such as
carcinogenesis, metastasis, anti-apoptosis, and cell proliferation, and
the resistance against anti cancer agent therapy (Non Patent Documents 5
and 6).

[0008] Accordingly, a compound inhibiting NFκB is useful as a
preventive or therapeutic agent for chronic inflammatory disease,
autoimmune disease, viral disease, immune disease, novel cancer therapy,
and other diseases attributable to the activation of NFκB, and such
a compound is actively developed.

[0009] Meanwhile, tylophorine represented by the following formula (A) and
an analog thereof are called phenanthroindolizidine alkaloid, which is a
compound mainly obtained from a plant belonging to the family
Asclepiadaceae (the genera Tylophora, Vincetoxicum, Pergularia, and
Cynanchum) (Non Patent Document 11).

[0010] Also, some of the aforementioned plants belonging to the genus
Tylophora are known as raw materials for anti-inflammatory drugs,
antiasthma drugs, and antiameba drugs (Non Patent Document 12). Also,
tylophorine is known to exhibit a potent cytotoxic activity, and a
research on the synthetic method thereof is also vigorously conducted
(Non Patent Document 13). Further, among the above-noted
phenanthroindolizidine alkaloid, tylocrebrine represented by the
following formula (B) is known to have neurotoxicity (Non Patent Document
14). Also, recently, it is known that tylophorine analogs represented by
the following formulas (C) and (D) have consistently exhibited a potent
cytotoxic activity in the NCI-60 tumor cell panel study, and that the
mechanism of action of those tylophorine analogs is different from that
of existing antitumor agents (Non Patent Document 15). Further, a
compound represented by the following formula (E), which is
phenanthroindolizidine alkaloid derived from the insect, is known to have
a potent cytotoxic activity (Non Patent Document 16).

[0011] Furthermore, phenanthroindolizidine alkaloid is known to inhibit
transcription mediated by NFκB, which is a transcription factor
(Non Patent Document 15).

[0029] Accordingly, it is an object of the present invention to provide a
novel compound having an excellent NFκB inhibitory action.

Means of Solving the Problem

[0030] Despite the fact that phenanthroindolizidine alkaloid has a potent
cytotoxic activity and an interesting mechanism of action as described
above, there are very few reports on the systemic and comprehensive
assessment of the biological activity, particularly the assessment of the
in vivo antitumor activity, of such alkaloid (Non Patent Documents 15 and
17).

[0031] Under such a circumstance, the present inventors conducted an
intensive research to achieve the aforementioned object. As a result,
they have found that a compound represented by the following formula (1)
or a salt thereof have excellent NFκB inhibitory action, antitumor
action, and anti-inflammatory action, while having few side effects and
excellent solubility, and thus is useful as a medicine such as an
anticancer agent, whereby completing the present invention.

[0032] That is, the present invention provides a compound represented by
the following formula (1) or a salt thereof:

##STR00003##

wherein, R1 represents a hydrogen atom, a lower alkyl group, a lower
alkyloxy group, or a halogen atom; R2 represents a hydroxyl group,
or a lower alkyloxy group; R3 represents a hydrogen atom, a lower
alkyl group, or a halogen atom; R4 represents a hydrogen atom or a
lower alkyloxy group; R5 represents a hydrogen atom, a lower
alkyloxy group, a halogen atom, a hydroxyl group, or a methylenedioxy
group formed together with R6 or an isopropylidenedioxy group formed
together with R6; R6 represents a hydrogen atom, a lower
alkyloxy group, or a methylenedioxy group formed together with R5 or
an isopropylidenedioxy group formed together with R5; R7
represents a hydrogen atom or a lower alkyl group; and R8 represents
a hydrogen atom, a hydroxyl group, an amino group, or a lower
alkylcarbonyloxy group; provided that the following cases are excluded:
the case where R1, R3, R4, and R7 are hydrogen atoms;
R2 and R8 are hydroxyl groups and R5 and R6 are
methoxy groups; the case where R1, R3, R4, and R7 are
hydrogen atoms, R2, R5, and R6 are methoxy groups, and
R8 is a hydroxyl group; the case where R1, R3, R4,
R7, and R8 are hydrogen atoms and R2, R5, and R6
are methoxy groups; the case where R1, R3, R4, and R7
are hydrogen atoms, R2, R5, and R8 are hydroxyl groups,
and R6 is a methoxy group; the case where R1, R3, R4,
and R7 are hydrogen atoms, R2 and R6 are methoxy groups,
and R5 and R8 are hydroxyl groups; the case where R1,
R3, R4, R7, and R8 are hydrogen atoms, R2 and
R6 are methoxy groups, and R5 is a hydroxyl group; the case
where R1, R2, R5, and R6 are methoxy groups and
R3, R4, R7, and R8 are hydrogen atoms; the case where
R1, R2, R4, and R5 are methoxy groups and R3,
R6, R7, and R8 are hydrogen atoms; the case where R1,
R2, R5, and R6 are methoxy groups, R3, R4, and
R7 are hydrogen atoms, and R8 is a hydroxyl group; the case
where R1 and R2 are methoxy groups, R3, R4, R6,
R7, and R8 are hydrogen atoms, and R5 is a hydroxyl group;
the case where R1, R2, and R5 are methoxy groups, and
R3, R4, R6, R7, and R8 are hydrogen atoms; the
case where R1, R2, and R5 are methoxy groups, R3,
R4, R6, and R7 are hydrogen atoms, and R8 is a
hydroxyl group; the case where R1, R2, and R5 are methoxy
groups, R3, R4, R6, and R8 are hydrogen atoms, and
R7 is a methyl group; the case where R1, R2, and R5
are methoxy groups, R3, R4, and R6 are hydrogen atoms,
R7 is a methyl group, and R8 is a hydroxyl group; the case
where R1, R2, and R6 are methoxy groups, R3, R4,
R7, and R8 are hydrogen atoms, and R5 is a hydroxyl group;
and the case where R1, R2, and R4 are methoxy groups,
R3, R6, R7, and R8 are hydrogen atoms, and R5 is
a hydroxyl group.

[0033] The present invention also provides a medicine containing a
compound represented by the above formula (1) or a salt thereof as an
active ingredient.

[0034] The present invention also provides a pharmaceutical composition
containing a compound represented by the above formula (1) or a salt
thereof and a pharmaceutically acceptable carrier.

[0035] The present invention further provides use of a compound
represented by the above formula (1) or a salt thereof for the production
of a medicine.

[0036] The present invention further provides a method for preventing or
treating diseases associated with accelerated NFκB activity, cancer
or inflammatory disease characterized by administering a compound
represented by the above formula (1) or a salt thereof.

EFFECTS OF THE INVENTION

[0037] The compound represented by the formula (1) or the salt thereof of
the present invention has excellent NFκB inhibitory action,
antitumor action, and anti-inflammatory action, while having few side
effects and excellent solubility, thus it is useful as a medicine, an
NFκB inhibitor, a preventive or therapeutic agent for diseases
associated with accelerated NFκB activity including anticancer
agents (proliferation or metastasis of cancer), resistance against
anticancer agents, inflammatory disease (rheumatoid arthritis,
osteoarthritis, atopic dermatitis, bronchial asthma, psoriasis,
inflammatory bowel disease, and the like), cardiovascular disease
(ischemic disease, vascular restenosis after percutaneous transluminal
coronary angioplasty (PTCA), and the like), pulmonary fibrosis, diabetes,
autoimmune disease, viral disease, Alzheimer's disease, ichorrhemia,
metabolic syndrome, and the like. More specifically, the compound of the
present invention represented by the formula (1) or the salt thereof is
useful as an anticancer agent and a preventive or therapeutic agent for
various inflammatory diseases.

BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1 is a graph showing the antitumor effect of the compound of
the present invention in mice transplanted with human promyelocytic
leukemia HL-60 cells.

[0039] FIG. 2 is a graph showing the anti-inflammatory effect of the
compound of the present invention in mice with TPA-induced ear edema.

MODE FOR CARRYING OUT THE INVENTION

[0040] In the general formula (1), examples of R1 include a hydrogen
atom, a lower alkyl group, a lower alkyloxy group, and a halogen atom, of
which a hydrogen atom, or the following functional groups are
particularly preferable.

[0041] Examples of the lower alkyl group include an alkyl group with a
carbon number of 1 to 6. Specific examples thereof include a methyl
group, an ethyl group, a propyl group, a butyl group, a pentyl group, and
a hexyl group. Among these, a methyl group is particularly preferable.

[0042] Examples of the lower alkyloxy group include an alkyloxy group with
a carbon number of 1 to 6. Specific examples thereof include a methoxy
group, an ethoxy group, a propoxy group, a butoxy group, a pentyloxy
group, and a hexyloxy group. Among these, a methoxy group is particularly
preferable.

[0043] Also, examples of the halogen atom include a chlorine atom, a
bromine atom, a fluorine atom, and an iodine atom. Among these, a
chlorine atom and a fluorine atom are particularly preferable.

[0044] That is, in the general formula (1), as R1, a hydrogen atom, a
methyl group, a methoxy group, a chlorine atom, or a fluorine atom is
particularly preferable.

[0045] In the general formula (1), examples of R2 include a hydroxyl
group and a lower alkyloxy group. Among these, a hydroxyl group or the
following functional groups are particularly preferable.

[0046] Examples of the lower alkyloxy group include an alkyloxy group with
a carbon number of 1 to 6. Specific examples thereof include a methoxy
group, an ethoxyl group, a propoxy group, a butoxy group, a pentyloxy
group, and a hexyloxy group. Among these, a methoxy group is particularly
preferable.

[0047] That is, in the general formula (1), as R2, a hydroxyl group
or a methoxy group is particularly preferable.

[0048] In the general formula (1), examples of R3 include a hydrogen
atom, a lower alkyl group, and a halogen atom. Among these, a hydrogen
atom or the following functional groups are particularly preferable.

[0049] Examples of the lower alkyl group include an alkyl group with a
carbon number of 1 to 6. Specific examples thereof include a methyl
group, an ethyl group, a propyl group, a butyl group, a pentyl group, and
a hexyl group. Among these, a methyl group is particularly preferable.

[0050] Also, examples of the halogen atom include a chlorine atom, a
bromine atom, a fluorine atom, and an iodine atom. Among these, a
chlorine atom or a fluorine atom is particularly preferable.

[0051] That is, in the general formula (1), as R3, a hydrogen atom, a
methyl group, a chlorine atom, or a fluorine atom is particularly
preferable.

[0052] In the general formula (1), examples of R4 include a hydrogen
atom and a lower alkyloxy group. Among these, a hydrogen atom or the
following functional groups are particularly preferable.

[0053] Examples of the lower alkyloxy group include an alkyloxy group with
a carbon number of 1 to 6. Specific examples thereof include a methoxy
group, an ethoxyl group, a propoxy group, a butoxy group, a pentyloxy
group, and a hexyloxy group. Among these, a methoxy group is particularly
preferable.

[0054] That is, in the general formula (1), as R4, a hydrogen atom or
a methoxy group is particularly preferable.

[0055] In the general formula (1), examples of R5 include a hydrogen
atom, a lower alkyloxy group, a halogen atom, a hydroxyl group, a
methylenedioxy group formed together with R6, and an
isopropylidenedioxy group formed together with R6. Among these, a
hydrogen atom, a hydroxyl group, a methylenedioxy group formed together
with R6, and an isopropylidenedioxy group formed together with
R6, or the following functional groups are particularly preferable.

[0056] Examples of the lower alkyloxy group include an alkyloxy group with
a carbon number of 1 to 6. Specific examples thereof include a methoxy
group, an ethoxyl group, a propoxy group, a butoxy group, a pentyloxy
group, and a hexyloxy group, and among these, a methoxy group and an
ethoxy group are particularly preferable.

[0057] Also, examples of the halogen atom include a chlorine atom, a
bromine atom, a fluorine atom, and an iodine atom, and among these, a
fluorine atom is particularly preferable.

[0058] That is, in the general formula (1), as R5, a hydrogen atom, a
methoxy group, an ethoxy group, a fluorine atom, a hydroxyl group, a
methylenedioxy group formed together with R6, or an
isopropylidenedioxy group formed together with R6 is particularly
preferable.

[0059] In the general formula (1), examples of R6 include a hydrogen
atom, a lower alkyloxy group, a methylenedioxy group formed together with
R5, or an isopropylidenedioxy group formed together with R5.
Among these, a hydrogen atom, a methylenedioxy group formed together with
R5, an isopropylidenedioxy group formed together with R5, or
the following functional groups are particularly preferable.

[0060] Examples of the lower alkyloxy group include an alkyloxy group with
a carbon number of 1 to 6. Specific examples thereof include a methoxy
group, an ethoxy group, a propoxy group, a butoxy group, a pentyloxy
group, and a hexyloxy group, and among these, a methoxy group and an
ethoxy group are particularly preferable.

[0061] That is, in the general formula (1), as R6, a hydrogen atom, a
methoxy group, an ethoxy group, a methylenedioxy group formed together
with R5, or an isopropylidenedioxy group formed together with
R5 is particularly preferable.

[0062] In the general formula (1), examples of R7 include a hydrogen
atom and a lower alkyl group, and among these, a hydrogen atom or the
following functional groups are particularly preferable.

[0063] Examples of the lower alkyl group include an alkyl group with a
carbon number of 1 to 6. Specific examples thereof include a methyl
group, an ethyl group, a propyl group, a butyl group, a pentyl group, and
a hexyl group, and among these, a methyl group is particularly
preferable.

[0064] That is, in the general formula (1), as R7, a hydrogen atom or
a methyl group is particularly preferable.

[0065] In the general formula (1), examples of R8 include a hydrogen
atom, a hydroxyl group, an amino group, and a lower alkylcarbonyloxy
group. Particularly, a hydrogen atom, a hydroxyl group, an amino group,
or the following functional groups are particularly preferable.

[0066] Examples of the lower alkylcarbonyloxy group include an
alkylcarbonyloxy group with a carbon number of 1 to 6. Specific examples
thereof include an acetoxy group, a propionyloxy group, and a butyryloxy
group, and among these, an acetoxy group is particularly preferable.

[0067] That is, in the general formula (1), as R8, a hydrogen atom, a
hydroxyl group, an amino group, or an acetoxy group is particularly
preferable.

[0068] A compound of the general formula (1), wherein R1 is a
hydrogen atom; R2 is a hydroxyl group; R3 is a hydrogen atom;
R4 is a hydrogen atom; R5 is a methoxy group; R6 is a
methoxy group; R7 is a hydrogen atom; and R8 is a hydrogen
atom, is more preferable.

[0069] In the present invention, the compound of the above formula (1) has
two stereocenters (carbon atoms at which R7 and R8 are
substituted). Because these stereocenters could take either an R
configuration or an S configuration, four kinds of stereoisomers are
possible. However, all of such stereoisomers and a mixture of various
combinations of stereoisomers are encompassed by the scope of the present
invention.

[0070] Examples of the isomer include (a configuration in which R7=S,
R8=S), (a configuration in which R7=R, R8=R), (a
configuration in which R7=S, R8=R), and (a configuration in
which R7=R, R8=S). Among these, (a configuration in which
R7=S, R8=S) is particularly preferable since a compound having
such a configuration strongly inhibits NFκB without inducing
unfavorable side effects.

[0071] In the present invention, a compound of the following formula (2)
or a pharmaceutically acceptable salt thereof is more preferable since
such a compound or salt strongly inhibits NFκB without inducing
unfavorable side effects.

##STR00004##

[0072] wherein, R1, R2, R3, R4, R5, R6,
R7, and R8 are the same as above.

[0073] When the compound represented by the formula (1) has a conformation
represented by the formula (2), the compound preferably has a hydrogen
atom at R8 since such a compound strongly inhibits NFκB
without inducing unfavorable side effects.

[0096] In the present invention, a salt of a compound represented by the
general formulas (1) or (2) may be a pharmaceutically acceptable salt.
Examples thereof include an inorganic acid salt such as hydrochloride,
sulfate, phosphate, hydrobromide, hydroiodide, nitrate, pyrosulfate, and
metaphosphate; an organic acid salt such as citrate, oxalate, benzoate,
acetate, trifluoroacetate, propionate, succinate, fumarate, lactate,
maleate, tartrate, glutarate, citrate, sulfonate (for example,
methanesulfonate, p-toluenesulfonate, and naphthalenesulfonate); and a
metal salt such as a lithium salt, a sodium salt, a potassium salt, a
magnesium salt, and a calcium salt.

[0097] The compound of the present invention can be produced, for example,
in accordance with the following reaction formula (a compound in which
R7=H, R8=OH (compound j) and a compound in which
R7=R8=H (compound 1) in the general formula (1) or (2) will be
shown as examples).

##STR00005## ##STR00006##

[0098] wherein, the groups R1 to R6 represent the same groups as
mentioned above, or if there is a functional group involved in the
reaction, such a group may be appropriately protected.

[0099] That is, benzaldehyde was reacted with benzyl cyanide to give a
compound (a), which was cyclized to give a compound (b). Subsequently, a
cyano group was reduced, whereby aldehyde (c) was obtained. After that, a
carbonyl group was reduced to give alcohol (d), which was brominated to
give (e), and then, the (e) was reacted with glutamic acid ester,
followed by cyclization, whereby (f) was obtained. The (f) was hydrolyzed
to give (g), from which (h) was obtained through intramolecular
acylation. After that, a carbonyl group was reduced to give (i), followed
by reduction of lactam, whereby phenanthroindolizidine (j) having a
hydroxyl group at R8 was obtained. The hydroxyl group at R8 of
the compound (i) was reductively removed to give (k), followed by
reduction of lactam, whereby phenanthroindolizidine (l) having a hydrogen
atom at R8 was obtained.

[0100] The reaction of benzaldehyde with benzyl cyanide is preferably
carried out in alcohol in the presence of a base. At this point, specific
examples of the base include sodium methoxide and sodium ethoxide.

[0101] The cyclization of the compound (a) is preferably carried out by
photoirradiation in the presence of iodine and propylene oxide. Also, a
cyclization reaction involving treatment with vanadium (V) or thallium
(III) may be employed.

[0102] The reduction of the compound (b) is preferably carried out by
reacting diisobutylaluminum hydride. Also, the reduction of the compound
(c) is preferably carried out by reacting sodium borohydride.

[0103] The bromination of the compound (d) is preferably carried out by
reacting phosphorous tribromide in the presence of triethylamine. Also,
the bromination may be carried out by allowing carbon tetrabromide to act
in the presence of triphenylphosphine.

[0104] The amination-lactamization of the compound (e) with L-glutamic
acid diisopropyl ester is preferably carried out in a solvent such as
dimethylformamide in the presence of a base such as potassium carbonate,
and allowing an acid such as acetic acid to act on the resulting aminated
product in alcohol such as methanol. At this point, when D-glutamic acid
diisopropyl ester is used, a corresponding enantiomer is obtained.

[0105] The hydrolysis of the compound (f) is preferably carried out using
a base in a solvent such as methanol. At this point, specific examples of
the base include potassium hydroxide and sodium hydroxide.

[0106] The intramolecular Friedel-Crafts reaction of the compound (g) is
preferably carried out in a solvent such as methylene chloride by
converting the compound (g) to acid chloride by oxalyl chloride within a
system, followed by treatment with a Lewis acid. At this point, specific
examples of the Lewis acid include tin chloride and aluminum chloride.

[0107] The reduction of the compound (h) is preferably carried out using a
reducing agent such as sodium borohydride and lithium tri-secondary butyl
borohydride. For stereoselective reduction, the reduction is preferably
carried out using a reducing agent such as lithium tri-secondary butyl
borohydride.

[0108] The reduction of the lactam of the compound (i) is preferably
carried out using a reducing agent such as borane and lithium aluminum
hydride.

[0109] The reduction of the hydroxyl group of the compound (i) is
preferably carried out by a combination of an acid and a reducing agent.
As the acid, trifluoroacetic acid, a boron trifluoride-diethyl ether
complex, and the like are preferable. As the reducing agent,
triethylsilane is preferable.

[0110] The reduction of the lactam of the compound (k) is preferably
carried out using a reducing agent such as borane and lithium aluminum
hydride.

[0111] As will be shown in the following Examples, a compound represented
by the formula (1) or a salt thereof have excellent NFκB inhibitory
action, antitumor action and anti-inflammatory action.

[0113] In the present invention, no particular limitation is imposed on
the "NFκB inhibitor" as long as it has an inhibitory action on
NFκB. More specifically, an NFκB inhibitor exhibits an
IC50 value of the inhibitory action on NFκB of preferably 2000
ng/mL or less, more preferably 500 ng/mL or less, and particular
preferably 100 ng/mL or less, as measured by the method of Example 2
described below.

[0114] Also, an NFκB inhibitor exhibits an IC50 value of the
inhibitory action on cancer proliferation of preferably 2000 ng/mL or
less, more preferably 500 ng/mL or less, and particular preferably 100
ng/mL or less, as measured by the method of Example 3 described below.

[0115] When a compound represented by the formula (1) or a salt thereof is
used as a medicine, one kind of the compound or the salt thereof may be
used alone or plural kinds thereof may be used in combination. Further, a
compound represented by the formula (1) or a salt thereof may also be
used in combination with other therapeutically advantageous compounds,
and the mechanism of action of these therapeutically advantageous
compounds may be the same as or different from that of the compound of
the present invention.

[0116] When the compound of the present invention is used as a medicine,
it can be administered in any dosage form. Examples thereof include an
orally administered agent such as a tablet, a capsule, a granule, a
sugar-coated tablet, a pill, a fine granule, powder, a dust formulation,
a sustained-release formulation, a suspension, an emulsion, syrup, an
emulsified formulation, a lyophilized preparation, a liquid, and an
elixir; and a parenterally administered agent including an injection such
as an intravenous injection, an intramuscular injection, a subcutaneous
injection, or a drip infusion, an external agent such as an endermic
liniment or a patch, a suppository, an infusion solution, a percutaneous
agent, a transmucosal agent, a nasal agent, an inhalant, a bolus, and the
like.

[0117] When the compound is used as a medicine, a preparation can be
produced by an ordinary method, in which the compound represented by the
formula (1) or the salt thereof of the present invention may be employed
alone or in combination with a pharmaceutically acceptable carrier.
Examples of the pharmaceutically acceptable carrier include an excipient,
a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter,
a corrigent, a colorant, a flavor, a diluent, a disinfecting agent, an
osmotic pressure adjuster, a pH adjuster, an emulsifying agent, a
preservative, a stabilizer, an absorption aid, an antioxidant, an
ultraviolet absorber, a humectant, a viscosity enhancer, a glazing agent,
an activity enhancer, an anti-inflammatory agent, a tonicity agent, a
soothing agent, and a flavoring agent.

[0124] When a medicine containing a compound represented by the formula
(1) or a salt thereof as an active ingredient is systemically
administered, a preferable dosage form is an injection or an orally
administered agent, and as the injection, an intravenous injection is
particularly preferable. In that case, the medicine can be administered
via other injection routes such as a subcutaneous, intramuscular, or
intraperitoneal injection, or the medicine can be administered
transmucosally or percutaneously using a penetrant such as bile salt or
fuchsin acid, or other surfactants. The aforementioned administration of
a pharmaceutical composition may be given locally or in the form of an
ointment, a paste, a gel, and the like.

[0125] The NFκB inhibitor of the present invention can be used not
only as the pharmaceutical products as described above but also as foods,
drinks, and the like. In that case, the phenanthroindolizidine alkaloid
compound or the salt thereof of the present invention may be contained in
foods and drinks as-is or together with various nutritional components.
The foods and drinks obtained in such a manner can be utilized as food
products for health use or foodstuff which are useful for improvement,
prevention, etc. of proliferation or metastasis of cancer, resistance
against anticancer agents, inflammatory disease (rheumatoid arthritis,
osteoarthritis, atopic dermatitis, bronchial asthma, psoriasis,
inflammatory bowel disease, and the like), cardiovascular disease
(ischemic disease, vascular restenosis after percutaneous transluminal
coronary angioplasty (PTCA), and the like), pulmonary fibrosis, diabetes,
autoimmune disease, viral disease, Alzheimer's disease, ichorrhemia,
metabolic syndrome, and the like. These foods and drinks or a container
thereof may display that the foods and drinks have the aforementioned
effects. Specifically, when the NFκB inhibitor of the present
invention is added to foods and drinks, they may be shaped into a form
suitable for ingestion, for example, a granule, a grain, a tablet, a
capsule, and a paste, by ordinary means using additives permitted for use
in foods and drinks, if desired. Also, the NFκB inhibitor of the
present invention may be added to various food products, for example, a
processed meat product such as ham and sausage, a processed seafood
product such as cooked minced fish or fish sausage, bread, confectionery,
butter, dry milk, and fermented foods and drinks, or the NFκB
inhibitor of the present invention may also be added to drinks such as
water, fruit juice, milk, a soft drink, and a tea drink. It is to be
noted that the foods and drinks also include feed for the animal.

[0126] Further, as the foods and drinks, fermented milk products such as
fermented milk, fermented bacterial drinks, fermented soymilk, fermented
fruit juice, and fermented vegetable juice containing the
phenanthroindolizidine alkaloid compound or a salt thereof as an active
ingredient are preferably employed. These fermented milk foods and drinks
may be produced by an ordinary method. For example, fermented milk is
obtained by inoculating lactic acid bacteria and bifidobacteria into a
sterilized milk medium and culturing them, and subjecting the resulting
product to homogenization treatment to give a fermented milk base.
Subsequently, a separately-prepared syrup solution and the
phenanthroindolizidine alkaloid compound or a salt thereof are added and
mixed, and the resulting product is homogenized using a homogenizer and
the like, and a flavor is further added to prepare the final product. The
fermented milk foods and drinks obtained in such a manner may also be
provided in the form of, for example, any of plain type, soft type,
fruit-flavored type, solid, and liquid products.

[0127] No strict limitation is imposed on the dosage amount of the
phenanthroindolizidine alkaloid compound or a salt thereof, which is the
active ingredient of the NFκB inhibitor of the present invention.
Because the effects achieved vary depending on various usage patterns
involving the subject of administration, indication, and the like, the
dosage amount is desirably determined for each case, and a dosage amount
of the phenanthroindolizidine alkaloid compound or a salt thereof is
preferably 1 mg to 10 g, more preferably 10 mg to 1 g, per day.

[0128] The NFκB inhibitor of the present invention can be applied to
all kinds of mammals including human.

EXAMPLES

[0129] As described hereinbelow, while the present invention will be
further described in detail with Examples, the present invention is not
limited thereto.

[0130] The phenanthroindolizidine alkaloid of the present invention was
synthesized in accordance with a reaction pathway including the following
steps 1 to 10. When any of the substituents represented by R needed to be
protected for the reaction to proceed, a suitable protecting group was
used to carry out the reaction.

##STR00007## ##STR00008## ##STR00009##

Synthesis Example 1

[0131] A compound having the following groups at R1 to R6 was
synthesized. The operations of the steps 1 to 10 will be described below.

[0132] In a round-bottom flask, 160 mg (2.4 mmol, 0.1 eq.) of sodium
ethoxide was added to a suspension of 5.0 g (24.1 mmol) of
3,4-5-trimethoxybenzyl cyanide and 5.1 g (24.1 mmol, 1.0 eq.) of
4-hydroxybenzaldehyde in 150 mL of ethanol under an argon atmosphere at
room temperature while stirring, and the resulting mixture was heated to
reflux (the oil bath temperature: 85° C.). After three hours, the
disappearance of the raw materials was confirmed, and the resulting
reaction liquid was cooled on ice to precipitate a solid. The solid was
then collected by suction filtration using a Buchner funnel and a
filtering flask, which was then washed with 100 mL of methanol twice. The
solid was dried under reduced pressure at 60° C. to give 9.3 g
(97%) of light yellow powder.

[0134] In a photoreaction container, argon was infused into a solution of
4.0 g (10.0 mmol) of stilbene in 7 L of acetonitrile at room temperature
while stirring. After 10 minutes, 2.5 g (10.0 mmol, 1.0 eq.) of iodine
and 28 mL (400.0 mmol, 40 eq.) of propylene oxide were added, followed by
irradiation of light at room temperature while stirring. After 72 hours
of irradiation, the disappearance of the raw materials was confirmed, and
the resulting reaction liquid was concentrated. The residual product was
dissolved in 500 mL of chloroform, followed by washing with 1 L of
saturated sodium thiosulfate and 500 mL of brine. The organic layer was
dried over magnesium sulfate, and then the solvent was distilled under
reduced pressure to give a solid. The solid was collected by suction
filtration using a Buchner funnel and a filtering flask, which was then
washed with 50 mL of methanol twice. The solid was dried under reduced
pressure at 60° C. to give 2.6 g (64%) of light brown powder.

[0136] In a round-bottom flask, a solution of 5.1 mL of 1.0 M
diisobutylaluminum hydride in methylene chloride (5.1 mmol, 1.3 eq.) was
added dropwise to a solution of 1.6 g (3.9 mmol) of cyanide in 200 mL of
methylene chloride under an argon atmosphere while stirring with cooling
on ice. During the dropwise addition, the mixture turned into a yellow
suspension. The suspension was stirred for one hour on ice, and then for
three hours at room temperature, and then the disappearance of the raw
materials was confirmed. The resulting reaction liquid was cooled on ice,
and 100 mL of 10% hydrochloric acid was slowly added to the liquid. The
reaction liquid turned into a suspension, which was dissolved in a
solution of chloroform-methanol=4:1. The organic layer was separated and
the aqueous layer was extracted with a solution of
chloroform-methanol=4:1. The organic layer was combined and the resulting
mixture was dried over magnesium sulfate. The solvent was then removed
under reduced pressure to give 1.4 g (90%) of a yellow solid.

[0138] In a round-bottom flask, 148 mg (3.9 mmol, 1.1 eq.) of sodium
borohydride was added to a suspension of 1.4 g (3.5 mmol) of aldehyde in
40 mL of methanol and 80 mL of 1,4-dioxane under an argon atmosphere
while stirring with cooling on ice. After one hour, the disappearance of
the raw materials was confirmed, and 100 mL of brine was added to the
resulting reaction liquid. Further, a solution of chloroform-methanol=4:1
was added to give a complete solution, and then, the organic layer was
separated and the aqueous layer was extracted with a solution of
chloroform-methanol=4:1. The organic layer was combined and the resulting
mixture was dried over magnesium sulfate. The solvent was then removed
under reduced pressure to give 1.5 g (quant) of a light brown solid.

[0140] In a recovery flask, 492 μL (3.5 mmol, 1 eq.) of triethylamine
was added to a suspension of 1.5 g (3.5 mmol) of alcohol in 50 mL of
chloroform under an argon atmosphere. And then, while stirring with
cooling on ice, 336 μL (3.5 mmol, 1.0 eq.) of phosphorus tribromide
was slowly added dropwise. After two hours, the disappearance of the raw
materials was confirmed, and 30 mL of water was slowly added dropwise to
precipitate a solid. After 30 minutes, the solid was dissolved in a
solution of chloroform-methanol=4:1. The organic layer was separated and
the aqueous layer was extracted with a solution of
chloroform-methanol=4:1. The organic layer was collected and dried over
magnesium sulfate, and then, the solvent was removed under reduced
pressure to give 1.6 g (95%) of the reaction product.

Step 6: Introduction of a Glutamic Acid Unit

[0141] In a round-bottom flask, 491 mg (2.1 mmol, 1.25 eq.) of L-glutamic
acid diisopropyl ester and 588 mg (4.3 mmol, 2.5 eq.) of potassium
carbonate were added to a solution of 783 mg (1.7 mmol) of bromide in 20
mL of DMF and 20 mL of benzene, followed by stirring while heating at
80° C. After two hours, the disappearance of the raw materials was
confirmed. The resulting reaction liquid was cooled on ice, and 100 mL of
water and 100 mL of brine were added to the liquid. Further, 200 mL of
ethyl acetate was added, and then the organic layer was washed with each
of a saturated aqueous solution of sodium bicarbonate and saturated
saline. The resulting solution was dried over magnesium sulfate, and the
solvent was removed under reduced pressure to give an aminated crude
product.

[0142] A solution of the crude product obtained in such a manner in 16 mL
of methanol, 16 mL of 1,4-dioxane, and 8 mL of acetic acid was stirred at
45° C. After 16 hours, the disappearance of the raw materials was
confirmed. The resulting reaction liquid was allowed to stand to cool,
and 100 mL of brine was added to the liquid. Further, a saturated aqueous
solution of sodium bicarbonate was gradually added to make the aqueous
layer weakly basic. The aqueous layer was extracted with ethyl acetate,
and the organic layer was dried over magnesium sulfate. The solvent was
then removed under reduced pressure. The residual product was purified by
silica gel column chromatography (hexane:ethyl acetate=1:1) to give 153
mg (15%) of a white solid.

Step 7: Hydrolysis of Pyroglutamic Acid Ester

[0143] In a round-bottom flask, an aqueous solution of potassium hydroxide
(KOH: 70 mg (1.2 mmol, 4.5 eq.), H2O:5 mL) was added to a solution
of 153 mg (0.3 mmol) of ester in 10 mL of methanol and 20 mL of
1,4-dioxane at room temperature while stirring. After one hour, the
disappearance of the raw materials was confirmed, and the solvent was
distilled under reduced pressure. To the remaining aqueous solution, 1
mol/L hydrochloric acid was added little by little while stirring with
cooling on ice to achieve a pH of 2 to 3 to precipitate a white solid.
The white solid was collected by suction filtration using a Buchner
funnel and a filtering flask, which was washed with 50 mL of purified
water twice. The receiver was replaced by another filtering flask, and
the solid was dissolved in a solution of chloroform-methanol=4:1. The
resulting solution was transferred to a separatory funnel, and the
organic layer was separated and dried over magnesium sulfate. Thereafter,
the solvent was removed under reduced pressure to give 114 mg (81%) of a
yellow to white solid. The results of the steps 5 to 7 will be described
below.

[0145] In a round-bottom flask, 41 μL (0.5 mmol, 2.0 eq.) of oxalyl
chloride and one drop of DMF were added to a suspension of 114 mg (0.2
mmol) of carboxylic acid in 20 mL of methylene chloride under an argon
atmosphere at room temperature while stirring. After one hour, 690 μL
(0.7 mmol, 3.0 eq.) of 1.0 M tin chloride (IV) in methylene chloride was
slowly added. Upon completion of the dropwise addition, the resulting
mixture was heated to reflux. After four hours, the disappearance of the
raw materials was confirmed. The resulting reaction mixture (a brown to
orange suspension) was cooled on ice, and 50 mL of 1 mol/L hydrochloric
acid was added, followed by stirring for 30 minutes. A solution of
chloroform-methanol=4:1 was added to turn the mixture into a solution,
and subsequently the organic layer was washed with each of 1 mol/L
hydrochloric acid, a saturated aqueous solution of sodium bicarbonate,
and brine. The organic layer was dried over magnesium sulfate and the
solvent was removed under reduced pressure. The residual product was
purified by silica gel column chromatography (chloroform-methanol=50:1)
to give 70 mg (60%) of a yellow solid.

[0146] In a round-bottom flask, 300 μl (2.0 eq.) of lithium
tri-secondary butyl borohydride (1.0 M solution in THF) was added to 20
mL (70 mg (0.15 mmol)) of Ketone in THF at -78° C. under an argon
atmosphere. After one hour, the disappearance of the raw materials was
confirmed, and saturated aqueous ammonium chloride was then added to the
resulting reaction liquid to quench the reaction. The aqueous layer was
extracted with ethyl acetate, and the organic layer was dried over
anhydrous magnesium sulfate. The solvent was removed under reduced
pressure, and the residual product was purified by silica gel column
chromatography (chloroform-methanol=100:1) to give 39 mg (52%) of a brown
solid. The results of the steps 8 and 9 will be described below.

[0148] In a round-bottom flask, 320 μL (0.32 mmol, 4.0 eq.) of 1.0 M
BH3.THF in THF was added dropwise to a solution of 40 mg (0.08 mmol)
of lactam in 30 mL of THF under an argon atmosphere while stirring with
cooling on ice. After two hours, the disappearance of the raw materials
was confirmed. The resulting reaction liquid was cooled on ice, and 49
μL (0.32 mmol, 4.0 eq.) of N,N,N',N'-tetramethylethylenediamine was
added to the liquid while stirring. After 16 hours, the disappearance of
an amine-borane complex was confirmed, and the solvent was removed under
reduced pressure, and then, the residual product was purified by silica
gel column chromatography (chloroform-methanol=50:1) to give 16 mg (38%)
of a white solid.

[0151] The reactions were carried out using D-glutamic acid isopropyl
ester instead of L-glutamic acid diisopropyl ester in the aforementioned
step 6, and then in the subsequent steps, the reactions were carried out
in the same manner as that mentioned above to give an enantiomer. The
following enantiomers were also produced in the same manner.

<Enantiomer> (Compound 2)

[0152] yield: 13%, 95.8% ee

Synthesis Example 2

[0153] A compound having the following groups at R1 to R6 was
synthesized. The operations and the yield of each operation are shown
below.

[0195] To a suspension of 25 mg (0.06 mmol) of the compound obtained by
the step 10 in 10 mL of methanol, 5 mg of 10% palladium on carbon was
added, followed by stirring under a hydrogen atmosphere. After 24 hours,
the disappearance of the raw materials was confirmed, and the palladium
on carbon was removed by filtration. The resulting filtrate was
evaporated under reduced pressure and the residual product was purified
by column chromatography (chloroform:methanol=40:1) to give 7 mg (28.6%)
of a white solid. [α]D28+124.0 (c=0.28, CHCl3)

[0237] Derivatives in which stilbene was synthesized by a method different
from that described above were synthesized through the synthetic pathway
shown in the following steps 12 to 17. The reactions after the step 17
were carried out under similar reaction conditions to those used for the
reactions after the step 5 described above.

##STR00010##

Synthesis Example 6

[0238] A compound having the following groups at R1 to R6 was
synthesized. The operation and the yield of each operation are shown
below.

[0239] In a round-bottom flask, 8.6 g (41.4 mmol) of
3,4-isopropylidenedioxyphenyl acetate, 13.2 g (62.2 mmol, 1.5 eq.) of
4-benzyloxybenzaldehyde, 8.6 mL (62.2 mmol, 1.5 eq.) of triethylamine,
and 20 mL (207 mmol, 5 eq.) of anhydrous acetic acid were added, followed
by stirring while heating. After six hours, the disappearance of the raw
materials was confirmed. The resulting reaction liquid was allowed to
stand to cool and water was then added to quench the reaction. Diluted
hydrochloric acid was added and the resulting liquid was then extracted
with ethyl acetate. The organic layer was dried over anhydrous magnesium
sulfate, and then the solvent was removed under reduced pressure. The
residual product was purified by column chromatography (hexane:ethyl
acetate=5:1→1:1) to give 7.5 g (44.8%) of an oily product.

[0241] In a round-bottom flask, 2.4 mL (27.5 mmol, 1.5 eq.) of oxalyl
chloride was added to a solution of 7.3 g (18.4 mmol) of carboxylic acid
in 70 mL of methylene chloride, which was the raw material, under an
argon atmosphere. After 30 minutes, the production of acid chloride was
confirmed, and then the reaction was cooled to 0° C., followed by
addition of 3 mL (73.6 mmol, 4 eq.) of methanol. After one hour, the
disappearance of the raw materials was confirmed, and brine was added to
the resulting reaction liquid. The aqueous layer was extracted with
chloroform, and the organic layer was collected and dried over anhydrous
magnesium sulfate. The solvent was then removed under reduced pressure to
give 8.8 g (quant) of a crude product.

[0243] In a photoreaction container, argon was infused into a solution of
8.7 g (18.4 mmol) of stilbene in 7 L of acetonitrile for approximately 10
minutes at room temperature while stirring. After 10 minutes, 4.7 g (18.4
mmol, 1.0 eq.) of iodine and 52 mL (736.0 mmol, 40 eq.) of propylene
oxide were added, followed by irradiation of light at room temperature
while stirring. After 72 hours of irradiation, the disappearance of the
raw materials was confirmed, and the resulting reaction liquid was
concentrated. The residual product was dissolved in 500 mL of chloroform,
followed by washing with 1 L of saturated sodium thiosulfate and 500 mL
of saturated saline. The organic layer was dried over magnesium sulfate,
and then the solvent was removed under reduced pressure to give a solid.
The solid was collected by suction filtration using a Buchner funnel and
a filtering flask, which was then washed with 50 mL of methanol twice.
The solid was then dried under reduced pressure at 60° C. to give
6.3 g (66.5%) of light brown powder.

[0245] Under an argon atmosphere, a solution of 9.6 mL of 1.0 M
diisobutylaluminum hydride in toluene (9.6 mmol, 4.4 eq.) was added
dropwise to a solution of 900 mg (2.2 mmol) of methyl ester in 20 mL of
methylene chloride in a round-bottom flask at 0° C. After one
hour, the disappearance of the raw materials was confirmed, and 1 M
hydrochloric acid was slowly added to quench the reaction. The aqueous
layer was extracted with chloroform, and the organic layer was dried over
anhydrous magnesium sulfate. The solvent was then removed under reduced
pressure to give 988 mg (quant) of a crude product.

[0262] To 2 g (4.8 mmol) of raw materials in a round-bottom flask, 20 mL
of 6 N hydrochloric acid and 20 mL of acetic acid were added, followed by
heating under reflux. After four hours, the disappearance of the raw
materials was confirmed. The resulting reaction liquid was cooled on ice,
and brine was added thereto. The aqueous layer was extracted with ethyl
acetate, and the organic layer was dried over anhydrous magnesium
sulfate. The solvent was then removed under reduced pressure. Chloroform
was added to the residual product to precipitate a solid, which was
collected by filtration using a Kiriyama funnel. The thus-obtained solid
was dried under reduced pressure to give 956 mg (52.9%) of a reaction
product.

[0264] In a round-bottom flask, 2.2 g (15.6 mmol, 6 eq.) of
K2CO3 and 1.2 mL (15.6 mmol, 6 eq.) of ethyl bromide were added
to a solution of 956 mg (2.6 mmol) of diol in 30 mL of acetone, followed
by heating under reflux. After 12 hours, the disappearance of the raw
materials was confirmed, and acetone was removed under reduced pressure.
And then, brine was added to the residual product. The aqueous layer was
extracted with ethyl acetate, and the organic layer was dried over
anhydrous magnesium sulfate. The solvent was removed under reduced
pressure, and the residual product was purified through column
chromatography (hexane:ethyl acetate=3:1) to give 654 mg (76.9%) of a
white solid.

[0282] Phenanthroindolizidine alkaloid having an alkylcarbonyloxy group at
R2 or R8 was synthesized by acylation of phenanthroindolizidine
alkaloid having a corresponding hydroxyl group. The synthetic pathway is
shown in the following steps 18 and 19.

##STR00011##

Synthesis Example 8

[0283] A compound in which R'' represents CH3, which is obtained
through the aforementioned steps 18 and 19, was synthesized. The
operation and the yield of each operation are shown below (compound 5).

Step 18: Diacylation of Hydroxyl Groups at R2 and R8

[0284] In a 100 mL round-bottom flask, triethylamine (1.4 mL, 40 eq.),
anhydrous acetic acid (0.95 mL, 40 eq.), and dimethylaminopyridine (3 mg,
0.1 eq.) were added to a suspension of raw materials (90 mg, 0.25 mmol)
in methylene chloride (15 mL) under an argon atmosphere while stirring
with cooling on ice, followed by stirring for six hours. The
disappearance of the raw materials was confirmed, and then the resulting
reaction liquid was concentrated and purified through column
chromatography (CHCl3 only) to give 47 mg (41.9%) of a light yellow
solid.

[0287] In a 100 mL round-bottom flask, sodium bicarbonate (24 mg, 1.1 eq.)
was added to a solution of diacetyl (114 mg, 0.26 mmol) in
methanol/tetrahydrofuran/water (1:1:1) (6 mL) while stirring with cooling
on ice. The disappearance of the raw materials was confirmed, and then
the resulting mixture was extracted with ethyl acetate-hexane. The
organic layer was dried over anhydrous magnesium sulfate, followed by
removal of the solvent. The residual product was purified through column
chromatography (CHCl3 only→CHCl3:MeOH=200:1) to give 83
mg (78.4%) of a yellow solid.

[0290] The synthetic method for a compound obtained by reductively
removing a hydroxyl group of the compound obtained by the step 9 (a
hydroxyl group at the position R8 in the general formula (1) or (2))
will be described. The synthesis was performed in accordance with the
steps 20 and 21 shown below.

##STR00012##

Synthesis Example 9

[0291] A compound having the following groups at R1 to R6 was
synthesized through steps 20 and 21. The operation and the yield of each
operation are shown below.

[0311] In a round-bottom flask, 293 μL (2.31 mmol, 1.5 eq.) of a boron
trifluoride.diethyl ether complex was added to a solution of 719 mg (1.54
mmol) of alcohol in 10 mL of methylene chloride at 0° C. under an
argon atmosphere. After five minutes, 984 μL (6.16 mmol, 4.0 eq.) of
triethylsilane was added. After four hours, the disappearance of the raw
materials was confirmed, and a solution of chloroform-methanol=4:1 was
added to give a complete solution. And then, the organic layer was
separated and the aqueous layer was extracted with a solution of
chloroform-methanol=4:1. The organic layer was combined and the resulting
mixture was dried over magnesium sulfate. The solvent was then removed
under reduced pressure, and the residual product was purified through
column chromatography (chloroform-methanol=50:1) to give 516 mg (74%) of
a white solid.

[0440] A compound having an amino group at the position R8 in the
general formula (1) or (2) (compound 7) was synthesized as follows.

##STR00013##

Step 22: Conversion of Ketone to Oxime

[0441] In a round-bottom flask, 1 mL of pyridine and 70 mg (1.0 mmol, 2.3
eq.) of hydroxylamine hydrochloride were added to a solution of 200 mg
(0.43 mmol) of raw materials in ethanol (20 mL) and 1,4-dioxane (50 mL),
and the resulting mixture was heated under reflux. After 72 hours, the
disappearance of the raw materials was confirmed, and water was added to
the resulting reaction liquid to quench the reaction. The aqueous layer
was extracted with ethyl acetate, and the organic layer was dried over
anhydrous magnesium sulfate. The solvent was then removed under reduced
pressure and the residual product was purified through column
chromatography (chloroform:methanol=50:1→40:1) to give 120 mg
(57.8%) of a target compound.

[0443] Under an argon atmosphere, a solution of 50 mg (0.11 mmol) of raw
materials in 20 mL of THF was added dropwise to a 440 μl of 1.0 M
solution of lithium aluminium hydride in THF (0.44 mmol, 4 eq.) in a
round-bottom flask while stirring with cooling on ice, and subsequently
the resulting mixture was heated under reflux. After two hours, the
disappearance of the raw materials was confirmed, and 0.25 mL of 15%
sodium hydroxide was added to quench the reaction. The aqueous layer was
extracted with ethyl acetate, and the organic layer was dried over
anhydrous magnesium sulfate. The solvent was then removed under reduced
pressure and the residual product was purified through column
chromatography (chloroform:methanol=50:1→20:1) to give 31 mg
(60.1%) of a target compound.

[0481] The phenanthroindolizidine alkaloid compound described above
exhibited good solubility in a solvent. Particularly, when its
methanesulfonate salt was dissolved in an aqueous solution of 5% glucose,
it exhibited a sufficient solubility for administration (>10 mg/ml).

[0482] The compounds synthesized as above were used for biological
activity tests in the form of an arbitrary salt. Specifically, the salts
used were as follows.

[0483] It is to be noted that because the compounds 16, 17, 18, 19 and 20,
and the aforementioned compounds 11, 13, 12, and 5 are each the same in
structure, but only differ in the kind of salt; therefore, the synthetic
method for the former compounds is in accordance with the aforementioned
synthetic method.

[0484] The action of the phenanthroindolizidine alkaloid compound of the
present invention on the NFκB activity was studied in a luciferase
assay. Human colon cancer SW480 cells were transfected with
pNFκB-Luc Plasmid (Stratagene), which is a reporter vector in which
a five-time tandem repeat of the NFκB responsive element (NRE) is
integrated into the upstream of the luciferase gene, using Lipofectamine
2000 (Invitrogen Corporation) in accordance with the attached operating
procedure. Subsequently, the cells were cultured in an RPMI1640 medium
containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and
0.5 mg/mL G418 to produce SW480 cells having the luciferase gene, the
expression of which is regulated by NRE, stably introduced therein
(SW480/NRE-Luc cells). Likewise, SW480 cells were transfected with
pGL3-Control Vector (Promega Corporation), which is a reporter vector in
which the SV40 promoter is integrated into the upstream of the luciferase
gene, to produce SW480 cells having the luciferase gene, the expression
of which is regulated by the SV40 promoter, stably introduced therein
(SW480/SV40-Luc cells). The SW480/NRE-Luc cells or the SW480/SV40-Luc
cells were suspended in an RPMI1640 medium containing 10% FBS, 100 U/mL
penicillin, and 100 μg/mL streptomycin (10% FBS/RPMI1640), and then
seeded in a 96-well microplate, followed by culturing under conditions of
5% CO2 and at 37° C. (5000 cells/well). After an overnight
culture, the compound of the present invention was added, followed by
culturing for one hour. Further, 50 ng/mL TNFα (Sigma-Aldrich
Corporation) was added, followed by culturing for four hours.
Subsequently, a Steady-Glo Luciferase Assay reagent (Promega Corporation)
was added, and the luminescent intensity was detected by SpectraMax M5e
(Molecular Devices, Inc.) to measure the intracellular luciferase
activity. It is to be noted that the action of the compound of the
present invention on the NFκB activity or the SV40 promoter
activity was shown as IC50 values (the concentration of a test
compound needed for 50% inhibition of the induction of the luciferase
expression). The results are shown below.

[0485] As shown above, the phenanthroindolizidine alkaloid compound of the
present invention exhibited a potent inhibitory activity on the
NFκB activity. While pyrrolidine dithiocarbamate (PDTC), which is
known to have an NFκB inhibitory activity, was used as a positive
control drug in the present experiment, all of the compounds of the
present invention studied exhibited a stronger NFκB inhibitory
activity than did PDTC. Meanwhile, it was shown that these compounds did
not affect the SV40 promoter activity, indicating that they specifically
acted on NFκB.

Example 3

Inhibitory Action on the Proliferation of Cancer Cell

[0486] The action of the phenanthroindolizidine alkaloid compound of the
present invention on the proliferation of human colon cancer SW480 cells,
HT-29 cells, and human non-small cell lung cancer A549 cells was studied.
The SW480 cells were suspended in a 10% FBS/RPMI1640 and then seeded in a
96-well microplate, followed by culturing in 5% CO2 at 37° C.
(2000 cells/well). The A549 cells and the HT-29 cells were each suspended
in a DMEM medium containing 10% FBS, 100 U/mL penicillin, and 100
μg/mL streptomycin (10% FBS/DMEM) and a DMEM F-12 HAM medium
containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin
(10% FBS/DMEM F12 HAM), and then seeded in 96-well microplates, followed
by culturing in 5% CO2 at 37° C. (1000 cells/well). After an
overnight culture, the compound of the present invention was added,
followed by further culturing for 48 hours (SW480 cells) and 96 hours
(A549 cells and HT-29 cells). After culturing, the number of viable cells
was counted using TetraColor ONE (Seikagaku Corporation) in accordance
with the attached operating procedure. The results were expressed as the
concentration of a test compound needed for 50% inhibition of the
proliferation of the cells (IC50).

[0488] The antitumor effect of the phenanthroindolizidine alkaloid
compound of the present invention in vivo was studied using mice
transplanted with mouse fibrosarcoma Meth A cells. Meth A cells were
transplanted subcutaneously in the inguinal region of male 7-week-old
BALB/c mice (2.5×105 cells/mouse). Subsequently, on days 1, 5,
and 9, the compound of the present invention was intravenously
administered. To a control group, physiological saline as a solvent, was
administered. On day 21 after the cell transplantation, tumor was excised
and measured for its weight, and subsequently a tumor growth-inhibition
rate IR (%) was obtained by the following formula.

Tumor growth-inhibition rate IR (%)=(1-the weight of the tumor in an
administration group/the weight of the tumor in a control
group)×100

[0491] The antitumor effect of the phenanthroindolizidine alkaloid
compound of the present invention in vivo was studied using mice
transplanted with human colon cancer HCT116 cells. HCT116 cells were
transplanted subcutaneously in the inguinal region of male 6-week-old
BALB/c nude mice (2×106 cells/mouse). On days 1 to 5 and on
days 8 to 12 after the time at which the estimated tumor volume obtained
by 1/2ab2 (a and b indicate the major axis and the minor axis of
tumor, respectively) reached approximately 100 mm3 (day 0), the
compound of the present invention was administered (intraperitoneal
administration). To a control group, a 5% glucose solution as a solvent,
was administered. On day 21, tumor was excised and measured for its
weight, and subsequently a tumor proliferation-inhibition rate IR (%) was
calculated. The results are shown below.

[0493] The antitumor effect of the phenanthroindolizidine alkaloid
compound of the present invention in vivo was studied using mice
transplanted with human promyelocytic leukemia HL-60 cells. HL-60 cells
were transplanted subcutaneously in the inguinal region of male
6-week-old BALB/c nude mice (2×106 cells/mouse). On days 1, 5,
and 9 after the time at which the estimated tumor volume reached
approximately 100 mm3 (day 0), the compound of the present invention
was intravenously administered. To a control group, a 5% glucose solution
as a solvent, was administered. On day 15, tumor was excised and measured
for its weight, and subsequently a tumor growth-inhibition rate IR (%)
was calculated. As a result, as shown in FIG. 1, the
phenanthroindolizidine alkaloid compound of the present invention was
shown to exhibit an antitumor effect in mice transplanted with human
promyelocytic leukemia HL-60 cells. The tumor growth-inhibition rates IR
(%) in the groups receiving total doses of 50 and 100 mg/kg of the
compound were each found to be 57.4% (p<0.01 vs. control group) and
96.8% (p<0.001 vs. control group), respectively.

Example 7

Anti-Inflammatory Effect in Mice with TPA-Induced Ear Edema

[0494] The anti-inflammatory effect of the phenanthroindolizidine alkaloid
compound of the present invention in vivo was studied using mice with
phorbol 12-myristate 13-acetate (TPA)-induced ear edema. Compound 11, a
compound of the present invention, was intraperitoneally administered to
male 6-week-old ICR mice, and after 30 minutes, TPA (Sigma-Aldrich
Corporation) dissolved in acetone was applied to the front and back of
the right ear of the mice (5 μg/ear). Four hours after application of
TPA, the thickness of the auricle was measured by a dial thickness gauge
(Ozaki Mfg Co. Ltd.). A control group was administered with a 5% glucose
solution as a solvent. As a result, as shown in FIG. 2, it was shown that
the compound 11, a phenanthroindolizidine alkaloid compound of the
present invention, exhibited an anti-inflammatory effect by inhibiting
TPA-induced ear edema in a dose-dependent manner.

Example 8

Animal Toxicity Test

[0495] In order to examine the toxicity of the phenanthroindolizidine
alkaloid compound of the present invention in animals, the compound of
the present invention was intravenously administered to mice transplanted
with mouse fibrosarcoma Meth A cells (total doses were 25 and 50 mg/kg)
on days 1, 5, and 9 after the day of transplantation (day 0), and its
effect on the survival of the mice was observed for three weeks from the
initiation of the administration. Also, the toxicity of known
phenanthroindolizidine alkaloid compounds in mice, namely
(12aS,13S)-6,7-dimethoxy-9,10,11,12,12a,13-hexahydro-9a-aza-cyclopenta[b]-
triphenylene-3,13-diol (known compound 1; refer to WO01/023384) and
(12aS,13S)-3,6,7-trimethoxy-9,10,11,12,12a,13-hexahydro-9a-aza-cyclopenta-
[b]triphenylene-13-ol (known compound 2; refer to Planta Med., 2002, 68:
186-188), was simultaneously studied. To a control group, a physiological
saline solution as a solvent, was administered. The results are shown in
the following tables.

[0496] As shown above, all the mice survived in a group administered with
the compound of the present invention. On the other hand, all the mice
died in a group administered with 50 mg/kg of the known
phenanthroindolizidine alkaloid compounds (known compounds 1 and 2).
Particularly with the known compound 2, some of the mice also died in a
group administered with 25 mg/kg of the compound. From the above results,
the phenanthroindolizidine alkaloid compound of the present invention was
shown to have reduced toxicity in animals compared to the known compounds
1 and 2.

Example 9

Production of Tablets

[0497] The components shown below were mixed and the resulting mixture was
tableted.